Toxoplasma gondii-derived heat shock protein HSP70 functions as a B cell mitogen

Abstract

We have investigated the role of Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) as a B cell mitogen by measuring proliferative responses in vitro. TgHSP70 induced prominent proliferative responses in murine B cells derived not only from T gondii-infected but also from uninfected mice. Nude mice responded to TgHSP70; however, severe combined immunodeficiency, RAG1-/- B6, and microMT mice failed to respond. B220+ spleen cells showed marked proliferation after stimulation with TgHSP70, but neither CD4+ nor CD8+ population responded. This unresponsiveness of CD4+ and CD8- T cells to TgHSP70 was antigen presenting cells independent. These data indicate that TgHSP70 induced the proliferation of B cells but not T cells. Polymyxin B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate TgHSP70-induced proliferation. C3H/HeN mice responded well to TgHSP70 stimulation; however, C3H/HeJ mice carrying a point mutation in the Toll-like receptor (TLR) 4 failed to respond. This indicates that TLR4 is required for TgHSP70-induced B cell activation. The involvement of TLR4 in the TgHSP70-induced proliferative responses of spleen cells was also shown by the use of TLR4-/- mice. But TgHSP70-induced, but not LPS-induced, spleen cell proliferation was observed in MyD88-/- mice, indicating that the MyD88 molecule was involved in LPS-induced proliferation but not in TgHSP70-induced proliferation.

Fig. 1.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of recombinant TgHSP70 and TgHSP30. Recombinant TgHSP70 (lane 1) and TgHSP30 (lane 2) were run on a 12% SDS-PAGE with a molecular size marker (M)

Fig. 2.

Dose-response analysis. Spleen cells of uninfected B6 mice were cultured with the indicated amounts of TgHSP70 (closed circle) or TgHSP30 (closed triangle). Data are expressed as Δcpm of [³H]-thymidine incorporation. Representative data of 2 independent experiments are shown. Cpm, counts per minute

Fig. 3.

Kinetic study of the proliferative responses of spleen cells to TgHSP70. 1 × 10⁵ spleen cells from Toxoplasma gondii–infected (A) or uninfected (B) B6 (a susceptible strain) and BALB/c (a resistant strain) mice were cultured with 3 μg/mL of either TgHSP70 or TgHSP30 and were harvested on days 1–7. Proliferative responses are expressed as Δcpm of [³H]-thymidine incorporation. Symbols: spleen cells of B6 mice cultured with TgHSP70 (closed circle with solid line) or TgHSP30 (closed triangle with solid line), spleen cells of BALB/c mice cultured with TgHSP70 (open circle with dotted line) or TgHSP30 (open triangle with dotted line). The data are representative of 5 independent experiments. Cpm, counts per minute

Fig. 4.

Fractionation of spleen cells. Whole spleen cells of uninfected B6 mice were fractionated to B220⁺, CD4⁺, and CD8⁺ populations by a positive sorting with microbeads conjugated with anti-B220, anti-CD4, or anti-CD8 mAb. (A) B220⁺ population was stained with PE-conjugated anti-CD90 mAb and FITC-conjugated anti-B220 mAb. (B) CD4⁺ and (C) CD8⁺ populations were stained with PE-conjugated anti-CD8 mAb and FITC-conjugated anti-CD4 mAb. The purity of B220⁺, CD4⁺, and CD8⁺ populations by flow cytometry analysis was 96.1%, 95.9%, and 93.3%, respectively. PE, phycoerythrin; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate

Fig. 5.

TgHSP70 induced proliferation of B cells but not T cells. 1 × 10⁵ whole spleen cells and fractionated B220⁺, CD4⁺, or CD8⁺ population were cultured for 3 days with the absence (open bars) or presence (striped bars) of TgHSP70. 1 × 10⁵ mitomycin C–treated spleen cells were added as APC to the culture in the absence (shaded bars) or presence (filled bars) of TgHSP70. Data are representative of 5 independent experiments and are expressed as cpm of [³H]-thymidine incorporation. APC, antigen presenting cells; cpm, counts per minute

Fig. 6.

TgHSP70-induced B cell proliferative responses were subsided by the elimination of MHC class II positive cells. 1 × 10⁵ of B220⁺ or CD90⁺ spleen cells of B6 mice were treated with either complement (C) alone (open bars) or anti-I-A^b mAb plus C (shaded bars) and were cultured without or with TgHSP70, LPS, or Con A. 1 × 10⁵ of APC were added back to the culture treated with either C alone (striped bars) or anti-I-A^b mAb plus C (filled bars). Data are representative of 3 independent experiments and are expressed as Δcpm of [³H]-thymidine incorporation. MHC, major histocompatibility complex; mAb, monoclonal antibody; LPS, lipopolysaccharide; Con A, Concanavarin A; cpm, counts per minute

Fig. 7.

TgHSP70-induced proliferative responses of spleen cells from various strains of mice. TgHSP70-induced (striped bars) proliferative responses of 1 × 10⁵ spleen cells from uninfected B6 WT mice, FcγR^−/− B6 mice, ICR nude mice, μMT mice, RAG1^−/− B6 mice, and SCID mice were compared with LPS-induced (filled bars) or Con A–induced (dotted bars) proliferative responses. Data are representative of at least 2 independent experiments and are expressed as Δcpm of [³H]-thymidine incorporation. SCID, severe combined immunodeficiency; LPS, lipopolysaccharide; Con A, Concanavarin A; cpm, counts per minute

Fig. 8.

Polymyxin B did not inhibit TgHSP70-induced proliferative responses. Polymyxin B was added at 10 μg/mL and 5 μg/mL in the culture of TgHSP70-induced (closed circle) or LPS-induced (open circle) spleen cell proliferation assay. Data are expressed as Δcpm of [³H]-thymidine incorporation. Representative data from 3 independent experiments are shown. Differences of mean values between control and groups were determined with the unpaired Student's t-test. *, P < 0.003; **, P < 0.002; cpm, counts per minute

Fig. 9.

TLR4 expression of TgHSP70-stimulated B cells. B220⁺ spleen cells of B6 and TLR4^−/− mice were cultured for 36 hours in the absence or presence of either LPS, TgHSP70, or TgHSP30. The mRNA expression of TLR4 on B220⁺ spleen cells of B6 (lanes 1–4) and TLR4^−/− (lanes 5–8) mice unstimulated (lanes 1 and 5) or stimulated with LPS (lanes 2 and 6), TgHSP70 (lanes 3 and 7), and TgHSP30 (lanes 4 and 8) was examined by RT-PCR. As an internal control, GAPDH mRNA was tested. LPS, lipopolysaccharide; mRNA, messenger ribonucleic acid; RT-PCR, reverse transcriptase–polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Fig. 10.

Involvement of TLR4 but not MyD88 molecules in TgHSP70-induced spleen cell proliferation. (A) TgHSP70-induced (striped bars) or LPS-induced (filled bars) proliferative responses of 1 × 10⁵ spleen cells from B6 WT, C3H/HeN, and C3H/HeJ mice were compared. (B) TgHSP70-induced (striped bars) or LPS-induced (filled bars) proliferative responses of 1 × 10⁵ spleen cells from TLR2^−/−, TLR4^−/−, and MyD88^−/− mice were compared with those of B6 WT mice. Differences between mean values were analyzed with the unpaired Student's t-test. P values less than 0.05 were considered statistically significant. Data are representative of 5 independent experiments and are expressed as Δcpm of [³H]-thymidine incorporation. LPS, lipopolysaccharide; cpm, counts per minute